This invention relates to the production of cellulase in plastids using genetic constructs that express a fusion protein including endogluconase E1 fused to a transit peptide. The fusion site is cleavable by a stromal processing peptidase.
Cellulose is made up of repeating D-glucose residues, linearly linked via xcex2-1,4-glycosidic bonds. There can be more than 10,000 glucoses per polymer chain. Multiple chains adhere to one another to produce a crystalline structure, which in plant cell walls is usually associated with hemicellulose and lignin producing a complex matrix (Tomme et al., 1995; Teeri, 1997). Cellulose is synthesized by cellulose synthase, an enzyme that has remained elusive in plants until recently when its gene was cloned from Arabidopsis (Arioli et al., 1998).
There are two basic kinds of cellulases required for cellulose degradation. The endocellulases (also called endoglucanases) cleave the polymer chains internally, whereas exocellulases (referred to as exoglucanases) cleave from the reducing and nonreducing ends of the molecule, most often generated by the action endocellulases. Hence, the endo- and exo-cellulases work synergistically, and together are required for degradation of cellulose to glucose and celliogiose. The latter is then further cleaved by xcex2-glucosidase. Hence, a mixture of at least these three enzymes is needed eventually for enzymatic hydrolysis of cellulose for biomass conversion.
Cellulases usually have a structure defined by three domains, although there is considerable variation (Tomme et al., 1995). These include a catalytic domain for glycosidic bond cleavage, a region often rich in proline/serine and threonine that serves as a linker, and a cellulose binding domain (CBD). Endoglucanase E1 from the bacterium Acidothermus cellulocystis is useful as a prototype to investigate whether cellulases can be imported into the chloroplast. Endoglucanase E1 was isolated from Yellowstone National Park (Mohagheghi et al., 1986; Tucker et al., 1989). It is a thermostable enzyme, which may favorably affect its recovery in an active form from transgenic plants. It is synthesized as a precursor with a signal peptide that directs it to the export pathway in bacteria. The mature enzyme is 521 amino acids (aa) in length. The crystal structure of the catalytic domain of xcx9c40 kD (358 aa) was recently described (Sakon et al., 1996). Its pro/thr/ser-rich linker is 60 aa, and the CBD is 104 aa. The properties of the CBD that confer its function are not well-characterized. Endoglucanase E1 belongs to family 5 of the cellulases, which is one of the largest classified. These enzymes share a common (xcex1/xcex2)8 folded structure with a similar catalytic mechanism, yet they have only 7 conserved residues (Henrissat et al., 1995; Sakon et al., 1996). Mutational analysis shows these are necessary for activity (Bortoli-Terman et al., 1995).
General features of the pathway of protein import into chloroplasts are as follows:. Chloroplasts are multifunctional organelles that carry out numerous important metabolic processes in addition to photosynthesis. They are needed for the synthesis of fatty acids (e.g., palmitic, oleic and linoleic acids), branched (e.g., leucine) and essential aromatic amino acids (phenylalanine, tyrosine, tryptophan), starch, phytohormones, tetrapyroroles, terpenoids, as well as being required for nitrogen and sulfur reduction. Although chloroplasts contain their own transcriptional and translational machineries, only a small fraction of chloroplast proteins are encoded by their own genomes. Instead, the majority of proteins are encoded in the nucleus and synthesized in the cytosol as precursors with an N-terminal transit peptide, which facilities posttranslational import. The pathway of protein import into the chloroplast (and other plastid-types in different tissues) can be divided into six basic steps:
1) precursor polypeptide synthesis in the cytosol,
2) targeting to the envelope,
3) receptor recognition,
4) translocation across the outer and inner membranes,
5) proteolytic processing of the precursor, and
6) folding, localization and assembly of mature proteins within the organelle (a step obviously complicated in itself).
Although an increasingly sophisticated outline of the pathway is emerging, the mechanism underlying each step is not fully understood.
This invention relates to the production of cellulase in plastids using genetic constructs that express a fusion protein including endogluconase E1 fused to a transit peptide. The fusion site is cleavable by a stromal processing peptidase.
The invention also relates to expression of the gene constructs and production of active endogluconase E1 in vivo and in vitro.
An aspect of the invention is gene constructs that code for a precursor fusion protein with endogluconase E1 fused to a transit peptide, and a site cleavable by a stromal processing peptidase (SPP) at the junction of the transit peptide and endogluconase E1. Endogluconase E1 from Acidothermus cellulocystis, a thermophilic bacterium, is sequestered in plastids in an active form. This facilitates the production of usable and relatively inexpensive amounts of cellulase in transgenic plants, which subsequently can be used for cellulose degradation and glucose fermentation to ethanol. Only low levels of cellulase are present in normal plants, compared to transgenic plants made to overexpress the xe2x80x9cforeignxe2x80x9d or heterologous enzyme using a strong gene promoter.
Plants can serve as storage depots for the overexpression and accumulation of valuable enzymes needed in biotechnology. The enzymes must be accumulated in a site where they will not damage the cell. In the present invention, large amounts of the cellulase are sequestered away from the cell wall itself, where the enzyme might cause a modified or defective phenotype. Furthermore, there are hundreds of plastids (chloroplasts) per cell available to accumulate large amounts of cellulase. Therefore, the chloroplasts may also be isolated to enrich production.
Sequestering of endogluconase E1 in plastids in an active form may be the best approach to produce cellulase in transgenic plants. An aspect of the invention is to generate large amounts of this enzyme by recombinant technology for the degradation of cellulose to fermentable sugars, eventually yielding ethanol as an alternative fuel to fossil fuels.
To successfully express and import xcex2-1,4-endoglucanase E1 peptide (E1) into the stroma of plants, special gene constructs needed to be created. Constructs that code for a precursor fusion protein with E1 fused to a transit peptide and a site cleavable by the stromal processing peptidase (SPP) were developed.
Transit peptides and passenger proteins (the proteins transported by the transit proteins) must not fold or aggregate into structures that interfere with efficient processing and import for embodiments of the present invention. That is, they should be compatible. To obtain these fusion proteins, E1 and its catalytic domain (E1CD) were fused to two different transit peptides (TP). Both TP used, ferredoxin (FD) and Rubisco activase (RCBA), were linked to the E1 (521 amino acids in length) and E1CD (359 amino acids in length) passenger proteins. This alteration of the fused protein changed the efficiency by which cleavage and importation of the fused peptide occurred. To further study the efficiency of proteolytic processing and import, the number of amino acids in a spacer region immediately following the cleavage site (between the TP and passenger protein) was altered. Constructs that were developed were fused via a spacer region of 1, 5, 10, or 15 amino acid residues. The spacer region is between the cleavage site and the beginning of the E1 amino acid sequence.
One particular array of fusion proteins was made using the transit peptide of ferredoxin (FD). An unexpected finding was that the efficiency of proteolytic processing and import was significantly affected by the number of amino acids immediately following the cleavage site, before the start of the E1 amino acid sequence. Therefore, it appears that the spacing at the transit peptide-E1 junction is critical. This conclusion is supported by the observation that an insertion after the cleavage site of the C-terminal 10 amino acids from the signal peptide of E1 which is normally synthesized as a precursor that is exported from Acidothermusxe2x80x94also promotes processing and import.
Another unexpected finding is that a fusion protein containing only the catalytic domain (CD) of E1 is more readily cleaved and imported than a comparable precursor with full length E1. Both the E1 and CD polypeptides are enzymatically active when synthesized in vitro.
Extending the results with the transit peptide of FD, the transit peptide of Rubisco activase (RBCA) was fused to E1 and to CD. In comparable constructs, each transit peptide conferred different properties. Nevertheless, precursors with the transit peptide of RBCA were also cleaved by SPP and imported into chloroplasts.
An aspect of the invention is a fusion protein that includes endogluconase E1 and a transit peptide. The endogluconase E1 and transit peptide are joined to form a junction site that is cleavable by a stromal processing peptidase (SPP) discovered by the inventors and originally designated xe2x80x9cCPExe2x80x9d (chloroplast processing enzyme) before its ability to cleave most precursors targeted to the chloroplast was established. Suitable transit peptides include the peptide that transports ferredoxin (FD) and the peptide that transports Rubisco activase (RBCA). The fusion protein may include only a part of endoglucanase E1, preferably the catalytic domain (CD).
Another aspect of the invention includes a plant transformed with a fusion protein of the present invention.
Another aspect of the invention includes a method of producing cellulase in the plastids of transgenic plants. The method includes the following steps:
(a) expressing active endogluconase E1 in the transgenic plant; and
(b) collecting cellulase made by the transgenic plant.
Other aspects are a genetic construct comprising cDNA encoding a fusion protein of the present invention. The nucleus contains the gene construct. The chloroplasts will contain active E1 enzyme, without a transit peptide,